Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

Abstract

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1-4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings. redox stress; nitric oxide; hypoxia; cell death; inflammation THE BCL-2/ADENOVIRUS EIB 19-kDa interacting protein 3 (BNIP3, formerly known as NIP3) was first identified in a yeast twohybrid screen as a protein capable of interacting with the adenovirus protein E1B 19 kDa, a homolog of Bcl-2 (4). BNIP3 is a membrane-associated protein localized to mitochondria and other cytoplasmic membrane structures and is widely expressed in a large number of mouse and human tissues (8). BNIP3 appears to have an overall resemblance to several BH3-containing Bcl-2 family proteins such as BIK, BID, HRK, and BAD, in which the BH3 domain plays an important role in the induction of apoptosis. However, BNIP3 has been implicated in both apoptotic and necrotic cell death (5). For instance, the expression of BNIP3 (both mRNA and protein) is induced and related to hypoxia-induced apoptosis in a number of human and animal cell lines, including CHO-K1 (Chinese hamster ovary), CV-1 (monkey kidney), Rat-1 (rat fibroblast), PAM212 (human epithelial), HepG2 (human hepatocellular carcinoma), and ECV-304 (human bladder carcinoma) (5). Moreover, BNIP3 and Nix [a BNIP3 homolog sharing both structural and functional similarity (7), also known as BNIP3L (30), BNIP3␣ (50), and B5 (35)] are the only members of the Bcl-2 family of apoptotic factors induced in response to hypoxia (5). On the other hand, BNI...

Mechanisms and Biology of B-Cell Leukemia/Lymphoma 2/Adenovirus E1B Interacting Protein 3 and Nip-Like Protein X

“…Consistent with the notion that there is selective pressure against BNIP3 and NIX expression in cancer, deficiency of either protein promotes the growth of breast or osteosarcoma cell lines, respectively, in mice (28,63). BNIP3 and NIX are also upregulated in the setting of ischemic injury, such as glaucoma, stroke, hemorrhagic shock, and myocardial infarction (2,22,66,111,112). Collectively, these studies make a case that BNIP3 and NIX are involved in disease in various clinical settings.…”

Characterization of DISC Formation and TNFR1 Translocation to Mitochondria in TNF-α–Treated Hepatocytes

“…Primary hepatocytes were harvested from male Sprague-Dawley rats (Harlan, Indianapolis, IN) as previously described in detail. 18 The cells were washed and further cultured in fresh medium containing 5% calf serum in various treatments of reagents as specifically indicated in the figure legends.…”

“…Immunoprecipitated proteins were eluted with 2ϫ SDS loading buffer, separated by SDS gels, and transferred onto a nitrocellulose membrane, and immunoblot [Western blot (WB)] was processed as described previously. 18 …”

Cyanide-Induced Apoptosis of Dopaminergic Cells Is Promoted by BNIP3 and Bax Modulation of Endoplasmic Reticulum-Mitochondrial Ca2+ Levels

“…Burton et al (2006) observed in glial and glioblastoma tumor cells that BNIP3 is expressed in the nucleus, and hypoxia stimulates BNIP3 translocation to cytosol and mitochondria to participate in execution of cell death. Similarly in normoxic hepatocytes, constitutive BNIP3 is localized to the nucleus and after hypoxia, BNIP3 levels increased in cyto- plasm (Metukuri et al, 2009). Sequestering BNIP3 in the nucleus blocks BNIP3's association with mitochondria and other organelles, thereby suppressing execution of cell death.…”